Implication des Rho GTPases dans la survie cellulaire et l'adhérence : caractérisation d'une nouvelle voie d'activation de la Kinase Akt par Rac

Les RhoGTPases régulent une multitude de fonctions cellulaires. Les travaux présentés dans cette thèse mettent en évidence les interrelations entre deux phénomènes majeurs contrôlés par cette classe de molécules que sont la régulation de l’organisation du cytosquelette d’actine et la survie cellulaire. L’adhérence à la matrice extracellulaire joue un rôle actif dans la survie cellulaire essentiellement grâce à la kinase Akt qui assure la protection contre l’apoptose. La 1ère partie de ce travail a permis de caractériser une nouvelle voie d’activation d’Akt par la GTPase Rac, sollicitée dans des situations où l’interaction avec la matrice est réduite. Mise en évidence dans des cellules non adhérentes, nos travaux montrent que cette voie est également opérationnelle dans des cellules adhérentes mises en suspension. Ces observations sont supportées par la mise en évidence, in vitro, d’un complexe contenant Akt et Rac activées dans les cellules en suspension. Dans la 2ème partie, nous avons exploré le rôle des RhoGTPases dans la morphologie de cellules leucémiques atypiques, qui présentent des prolongements cytoplasmiques caractéristiques, les Tricholeucocytes. Nous démontrons une relation entre cette morphologie et un fort taux d’activation des GTPases Rac et Cdc42. Le traitement de ces cellules à l’IFN normalise ces activités par un mécanisme faisant intervenir RhoA et p53. Dans la 3ème partie, nous nous sommes intéressés à des structures d’adhérence appelés podosomes dont la formation est dépendante des GTPases Cdc42 et RhoA. Nous avons montré pour la première fois, l’existence de ces structures in vivo.RhoGTPases control a multitude of cellular functions. The work presented in this thesis highlights the relationships between two major phenomena controlled by this class of molecules which are the regulation of the organization of the actin cytoskeleton and cellular survival. Adhesion to the extracellular matrix plays an essential role in cellular survival thanks to the Akt kinase which ensures protection against the apoptose. The first part of this work led us to characterize a new pathway of activation of Akt by the GTPase Rac, requested when the interactions with the matrix are reduced. Highlighted in nonadherent cells, our work shows that this way is also operational in adherent cells grown in suspension. These observations are supported by the description, in vitro, of a complex containing Akt and Rac activated in the cells in suspension. In the 2nd part, we explored the role of RhoGTPases in the morphology of atypical leukemic cells, the Tricholeucocytes which present characteristic cytoplasmic protrusions. We show a relation between this morphology and a strong rate of activation of GTPases Rac and Cdc42. Treatment of these cells by IFN normalize the activities by a mechanism RhoA and p53 dependant. In the 3rd part, we were interested in structures of adherence called podosomes on which formation is dependent on GTPases Cdc42 and RhoA. We showed for the first time, the existence of these structures in vivo

Human Immunodeficiency Virus Type 1 Vif causes dysfunction of Cdk1 and CyclinB1: implications for cell cycle arrest

The two major cytopathic factors in human immunodeficiency virus type 1 (HIV-1), the accessory proteins viral infectivity factor (Vif) and viral protein R (Vpr), inhibit cell-cycle progression at the G2 phase of the cell cycle. Although Vpr-induced blockade and the associated T-cell death have been well studied, the molecular mechanism of G2 arrest by Vif remains undefined. To elucidate how Vif induces arrest, we infected synchronized Jurkat T-cells and examined the effect of Vif on the activation of Cdk1 and CyclinB1, the chief cell-cycle factors for the G2 to M phase transition. We found that the characteristic dephosphorylation of an inhibitory phosphate on Cdk1 did not occur in infected cells expressing Vif. In addition, the nuclear translocation of Cdk1 and CyclinB1 was disregulated. Finally, Vif-induced cell cycle arrest was correlated with proviral expression of Vif. Taken together, our results suggest that Vif impairs mitotic entry by interfering with Cdk1-CyclinB1 activation

CytoBinning:Immunological insights from multi-dimensional data

<div><p>New cytometric techniques continue to push the boundaries of multi-parameter quantitative data acquisition at the single-cell level particularly in immunology and medicine. Sophisticated analysis methods for such ever higher dimensional datasets are rapidly emerging, with advanced data representations and dimensional reduction approaches. However, these are not yet standardized and clinical scientists and cell biologists are not yet experienced in their interpretation. More fundamentally their range of statistical validity is not yet fully established. We therefore propose a new method for the automated and unbiased analysis of high-dimensional single cell datasets that is simple and robust, with the goal of reducing this complex information into a familiar 2D scatter plot representation that is of immediate utility to a range of biomedical and clinical settings. Using publicly available flow cytometry and mass cytometry datasets we demonstrate that this method (termed CytoBinning), recapitulates the results of traditional manual cytometric analyses and leads to new and testable hypotheses.</p></div

The Necrotic Signal Induced by Mycophenolic Acid Overcomes Apoptosis-Resistance in Tumor Cells

The amount of inosine monophosphate dehydrogenase (IMPDH), a pivotal enzyme for the biosynthesis of the guanosine tri-phosphate (GTP), is frequently increased in tumor cells. The anti-viral agent ribavirin and the immunosuppressant mycophenolic acid (MPA) are potent inhibitors of IMPDH. We recently showed that IMPDH inhibition led to a necrotic signal requiring the activation of Cdc42.Herein, we strengthened the essential role played by this small GTPase in the necrotic signal by silencing Cdc42 and by the ectopic expression of a constitutive active mutant of Cdc42. Since resistance to apoptosis is an essential step for the tumorigenesis process, we next examined the effect of the MPA–mediated necrotic signal on different tumor cells demonstrating various mechanisms of resistance to apoptosis (Bcl2-, HSP70-, Lyn-, BCR-ABL–overexpressing cells). All tested cells remained sensitive to MPA–mediated necrotic signal. Furthermore, inhibition of IMPDH activity in Chronic Lymphocytic Leukemia cells was significantly more efficient at eliminating malignant cells than apoptotic inducers.These findings indicate that necrosis and apoptosis are split signals that share few if any common hub of signaling. In addition, the necrotic signaling pathway induced by depletion of the cellular amount of GTP/GDP would be of great interest to eliminate apoptotic-resistant tumor cells

Exposed Hydrophobic Residues in Human Immunodeficiency Virus Type 1 Vpr Helix-1 Are Important for Cell Cycle Arrest and Cell Death

The human immunodeficiency virus type 1 (HIV-1) accessory protein viral protein R (Vpr) is a major determinant for virus-induced G2/M cell cycle arrest and cytopathicity. Vpr is thought to perform these functions through the interaction with partner proteins. The NMR structure of Vpr revealed solvent exposed hydrophobic amino acids along helices 1 and 3 of Vpr, which could be putative protein binding domains. We previously showed that the hydrophobic patch along helix-3 was important for G2/M blockade and cytopathicity. Mutations of the exposed hydrophobic residues along helix-1 were found to reduce Vpr-induced cell cycle arrest and cell death as well. The levels of toxicity during virion delivery of Vpr correlated with G2/M arrest. Thus, the exposed hydrophobic amino acids in the amino-terminal helix-1 are important for the cell cycle arrest and cytopathicity functions of Vpr

The Naturally Processed CD95L Elicits a c-Yes/Calcium/PI3K-Driven Cell Migration Pathway

Patients affected by chronic inflammatory disorders display high amounts of soluble CD95L. This homotrimeric ligand arises from the cleavage by metalloproteases of its membrane-bound counterpart, a strong apoptotic inducer. In contrast, the naturally processed CD95L is viewed as an apoptotic antagonist competing with its membrane counterpart for binding to CD95. Recent reports pinpointed that activation of CD95 may attract myeloid and tumoral cells, which display resistance to the CD95-mediated apoptotic signal. However, all these studies were performed using chimeric CD95Ls (oligomerized forms), which behave as the membrane-bound ligand and not as the naturally processed CD95L. Herein, we examine the biological effects of the metalloprotease-cleaved CD95L on CD95-sensitive activated T-lymphocytes. We demonstrate that cleaved CD95L (cl-CD95L), found increased in sera of systemic lupus erythematosus (SLE) patients as compared to that of healthy individuals, promotes the formation of migrating pseudopods at the leading edge of which the death receptor CD95 is capped (confocal microscopy). Using different migration assays (wound healing/Boyden Chamber/endothelial transmigration), we uncover that cl-CD95L promotes cell migration through a c-yes/Ca2+/PI3K-driven signaling pathway, which relies on the formation of a CD95-containing complex designated the MISC for Motility-Inducing Signaling Complex. These findings revisit the role of the metalloprotease-cleaved CD95L and emphasize that the increase in cl-CD95L observed in patients affected by chronic inflammatory disorders may fuel the local or systemic tissue damage by promoting tissue-filtration of immune cells

Implication des Rho GTPases dans la survie cellulaire et l'adhérence (caractérisation d'une nouvelle voie d'activation de la kinase Akt par Rac)

Les RhoGTPases régulent une multitude de fonctions cellulaires. Les travaux présentés dans cette thèse mettent en évidence les interrelations entre deux phénomènes majeurs contrôlés par cette classe de molécules que sont la régulation de l organisation du cytosquelette d actine et la survie cellulaire. L adhérence à la matrice extracellulaire joue un rôle actif dans la survie cellulaire essentiellement grâce à la kinase Akt qui assure la protection contre l apoptose. La 1ère partie de ce travail a permis de caractériser une nouvelle voie d activation d Akt par la GTPase Rac, sollicitée dans des situations où l interaction avec la matrice est réduite. Mise en évidence dans des cellules non adhérentes, nos travaux montrent que cette voie est également opérationnelle dans des cellules adhérentes mises en suspension. Ces observations sont supportées par la mise en évidence, in vitro, d un complexe contenant Akt et Rac activées dans les cellules en suspension. Dans la 2ème partie, nous avons exploré le rôle des RhoGTPases dans la morphologie de cellules leucémiques atypiques, qui présentent des prolongements cytoplasmiques caractéristiques, les Tricholeucocytes. Nous démontrons une relation entre cette morphologie et un fort taux d activation des GTPases Rac et Cdc42. Le traitement de ces cellules à l IFNa normalise ces activités par un mécanisme faisant intervenir RhoA et p53. Dans la 3ème partie, nous nous sommes intéressés à des structures d adhérence appelés podosomes dont la formation est dépendante des GTPases Cdc42 et RhoA. Nous avons montré pour la première fois, l existence de ces structures in vivo.BORDEAUX1-BU Sciences-Talence (335222101) / SudocSudocFranceF

Hydrophobic residues on Vpr helix-1 are important for virion-delivered Vpr cell cycle arrest.

<p>WT or mutant Vpr proteins (denoted by the single letter amino acid changes) were delivered (Vpr_v) into Jurkat cells. Virions containing WT Vpr were titrated (md, medium) so that a matched Vpr protein control could be compared to the mutants. (<b>A</b>) Western blot of the Jurkat cells for WT and mutant virion-delivered Vpr (bottom). β-actin is shown as a protein loading control (top). (<b>B</b>) Histograms of cell cycle analysis at 41 hr post-infection show DNA content of PI-stained cells by flow cytometry. All samples represent 10,000 cellular events. G1 and G2/M populations were modeled using the Watson Pragmatic cell cycle model, and the G2,M/G1 ratio in each infection is shown. The data are representative of three experiments.</p

Hydrophobic residues on Vpr helix-1 are important for incorporation into virions.

<p>(<b>A</b>) 293T cells were co-transfected with pcDNA3-hVpr plasmids expressing WT or mutant Vpr (or the empty vector control), pLVSV-G, and pNL4-3_{e-n-GFP RTm, VprΔ22-86} to produce virus for virion delivery of Vpr. At day 2 the virus stocks were harvested and the 293T cells were lysed. Western blot indicates protein levels for WT or mutant Vpr (bottom). A western blot for β-actin is used as a loading control (top). (<b>B</b>) Lysates were prepared from the virus stocks in (A). Viral lysates were analyzed for protein levels of WT or mutant Vpr by western blotting as in (A) (bottom). The HIV-1 p24 capsid (p24 CA) is shown as a protein loading control (top). (<b>C</b>) Densitometry of all bands in (B) was performed. The intensity of the Vpr protein band was normalized to p24 CA and plotted as % Vpr incorporation relative to WT Vpr for each mutant. The data are shown as the mean ± the standard deviation of three independent experiments.</p

Vpr G2/M arrest correlates with cell death induced by virion delivery.

<p>(<b>A</b>) WT or mutant Vpr proteins (denoted by the single letter amino acid changes) were delivered (Vpr_v) into Jurkat cells, and separately Jurkat cells were co-transfected with pcDNA3-hVpr plasmids expressing WT or mutant Vpr (or the empty vector control) and pEGFP-N1 as a transfection marker at a ratio of 5∶1. Infected and transfected cells were analyzed for DNA content of the PI-stained cells by flow cytometry. G1 and G2/M populations were modeled using the Watson Pragmatic cell cycle model, and the G2,M/G1 ratios were plotted on the x-axis. (<b>B</b>) The viability of the Vpr_v-treated cells was monitored over time by flow cytometry (detection of PI-negative, forward-scatter high events), and the percentage of viable cells is plotted over time. These data are representative of three experiments. (<b>C</b>) The G2,M/G1 ratios and viability of Vpr_v-treated cells at 165 hour post-infection from three independent experiments were plotted. Each measurement is color-coded as in (B). Spearman's rank test was used to determine the correlation.</p